A female subject inherits two X-chromosomes, one from each parent. Shortly after conception, each somatic cell randomly inactivates (turns off) one of the X chromosomes. All daughter cells arising subsequently will therefore carry the same pair of inactive and active X chromosome. Since the process is random, the likelihood is that there will be a roughly equal split between inactivation of the paternal or maternal X chromosome.
The process by which this inactivation occurs is by methylation of the genes present on the inactivated X chromosome. Methylation silences the gene, an example of epigenetic modification.
The phenomenon of random X-inactivation can be used to determine the clonality of haematopoetic cells, when these cells are suspected to be neoplastic.
Determining clonality is easy for B cells and T cells. Each B cell experiences an isotype switch from IgM to IgG, IgA, or IgE during its development. B cells then rearrange their immunoglobulin heavy chains and express only the kappa or lambda light chain. Thus a lymphoma arising from a B cell clone will carry a rearranged mu, gamma, alpha or epsilon heavy chain and either kappa or lambda light chain.
T cells don't express cell surface immunoglobulins, but do carry T cell receptors. Just like B cells, they rearrange these receptors upon exposure to their cognate antigen. T cells carry alpha/beta and gamma/delta receptors, although only one of these pairs is expressed. It is easier to test for rearrangement of gamma receptors, because they have less polymorphisms.
However, such unique receptors or cell surface immunoglobulins do not exist for other haematopoetic cells, such as natural killer cells, macrophages or eosinophils. (Yes, they do express generic receptors, but these are not unique for each cell, unlike in T or B cells, which have to recognise specific antigens)
In females, as long there is heterozygosity, inactivation of a gene present on the X chromosome can be used to determine clonality in these haematopoetic cells. Such genes include phosphoglycerate kinase (PGK), hypoxanthine guanine phosphoribosyl transferase and human androgen receptor. Such analysis cannot be done if a both alleles of the gene are the same, i.e. with a state of homozygosity.
PGK has been used, but carries the drawback that there is only 40% heterozygosity in the population for this gene. Most such analyses are now done with the Human Androgen Receptor gene (HUMARA), as the prevalence of heterozygosity is very high- around 90%.
Restriction endonucleases which are methylation sensitive, are used to digest the DNA on each allele. The methylated (inactivated) allele will not be broken down, while the unmethylated (active) allele will be. All daughter cells arising from a single (clonal) cell will carry either the unmethylated (cleaved) or methylated (uncleaved) DNA, but not both. In homozygotes, since the paternally and maternally inherited alleles would be identical, it would not be possible to tell whether the paternal or maternal allele of the gene was present in the cells under consideration.
Males carry only one X-chromosome, hence the phenomenon of random X inactivation cannot be used to determine clonality in men.
The process by which this inactivation occurs is by methylation of the genes present on the inactivated X chromosome. Methylation silences the gene, an example of epigenetic modification.
The phenomenon of random X-inactivation can be used to determine the clonality of haematopoetic cells, when these cells are suspected to be neoplastic.
Determining clonality is easy for B cells and T cells. Each B cell experiences an isotype switch from IgM to IgG, IgA, or IgE during its development. B cells then rearrange their immunoglobulin heavy chains and express only the kappa or lambda light chain. Thus a lymphoma arising from a B cell clone will carry a rearranged mu, gamma, alpha or epsilon heavy chain and either kappa or lambda light chain.
T cells don't express cell surface immunoglobulins, but do carry T cell receptors. Just like B cells, they rearrange these receptors upon exposure to their cognate antigen. T cells carry alpha/beta and gamma/delta receptors, although only one of these pairs is expressed. It is easier to test for rearrangement of gamma receptors, because they have less polymorphisms.
However, such unique receptors or cell surface immunoglobulins do not exist for other haematopoetic cells, such as natural killer cells, macrophages or eosinophils. (Yes, they do express generic receptors, but these are not unique for each cell, unlike in T or B cells, which have to recognise specific antigens)
In females, as long there is heterozygosity, inactivation of a gene present on the X chromosome can be used to determine clonality in these haematopoetic cells. Such genes include phosphoglycerate kinase (PGK), hypoxanthine guanine phosphoribosyl transferase and human androgen receptor. Such analysis cannot be done if a both alleles of the gene are the same, i.e. with a state of homozygosity.
PGK has been used, but carries the drawback that there is only 40% heterozygosity in the population for this gene. Most such analyses are now done with the Human Androgen Receptor gene (HUMARA), as the prevalence of heterozygosity is very high- around 90%.
Restriction endonucleases which are methylation sensitive, are used to digest the DNA on each allele. The methylated (inactivated) allele will not be broken down, while the unmethylated (active) allele will be. All daughter cells arising from a single (clonal) cell will carry either the unmethylated (cleaved) or methylated (uncleaved) DNA, but not both. In homozygotes, since the paternally and maternally inherited alleles would be identical, it would not be possible to tell whether the paternal or maternal allele of the gene was present in the cells under consideration.
Males carry only one X-chromosome, hence the phenomenon of random X inactivation cannot be used to determine clonality in men.
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